This invention relates to nucleic acid molecules useful for producing plants having enhanced pathogen resistance characteristics, transgenic plants expressing these nucleic acid molecules, methods of making such plants, and methods for increasing the resistance of plants to pathogens.
Plants are hosts to thousands of infectious diseases caused by a vast array of phytopathogenic fungi, bacteria, viruses, and nematodes. These pathogens are responsible for significant crop losses worldwide, resulting from both infection of growing plants and destruction of harvested crops (Baker et al., 1997).
Plants recognize and resist many invading phytopathogens by inducing a rapid defense response, termed the hypersensitive response (HR). HR results in localized cell and tissue death at the site of infection, which constrains further spread of the infection. This local response often triggers non-specific resistance throughout the plant, a phenomenon known as systemic acquired resistance (SAR). Once triggered, SAR provides resistance for days to a wide range of pathogens. The generation of the HR and SAR in a plant depends upon the interaction between a dominant or semi-dominant resistance (R) gene product in the plant and a corresponding dominant avirulence (Avr) gene product expressed by the invading phytopathogen. It has been proposed that phytopathogen Avr products function as ligands, and that plant R products function as receptors. Thus, in the widely accepted model of phytopathogen/plant interaction, binding of the Avr product of an invading pathogen to a corresponding R product in the plant initiates the chain of events within the plant that produces HR and SAR and ultimately leads to disease resistance. A detailed review of the current understanding of Avr/R gene product interactions is presented in Baker et al. (1997).
Because the systemic acquired resistance (SAR) response acts nonspecifically throughout the plant to provide enhanced resistance to many pathogens, it has been extensively studied as a possible mechanism for conferring broad-spectrum pathogen resistance. The SAR response is characterized by the induction of at least nine gene families (known as SAR genes) in uninfected leaves of the plant. Some of the SAR genes encode proteins that have antimicrobial activity, including glucanases, chitinases and the pathogenesis-related (PR) proteins such as PR-1. A number of chemicals are known to trigger SAR, including salicylic acid, 2,6-dichloroisonicotinic acid (INA), 1,2,3-benzothiadiazole-7-thiocarboxylic acid-S-methyl ester, and arachidonic acid when applied through the roots, sprayed onto leaves or injected into stems. These and other chemicals are being investigated as candidate agents for inducing SAR in crop plants on an agricultural scale. For a more detailed discussion of the SAR response, see Agrios (1997), Chapter 5.
While efforts to utilize the SAR response to enhance resistance to phytopathogens have largely been based on the external application of inducing agents, molecular genetic research has focused on isolating and manipulating plant resistance (R) genes. Since the cloning of the first R gene, Pto from tomato, which confers resistance to Pseudomonas syringae pv. tomato (Martin et al., 1993), a number of other R genes have been reported (for reviews see Hammond-Kosack and Jones, 1997 and Baker et al., 1997). Sequence analyses of the proteins encoded by these R genes have revealed a number of motifs that are conserved between various R proteins; including leucine rich repeats (LRR), nucleotide binding site (NBS), Toll-IL-1R homology (TIR), leucine zipper (LZ) and transmembrane (TM) domains (Baker et al., 1997).
Much effort is currently being directed towards using R genes to engineer pathogen resistance in plants. The production of transgenic plants carrying a heterologous gene sequence is now routinely practiced by plant molecular biologists, and methods for incorporating an isolated gene (such as an R gene) into an expression cassette, producing plant transformation vectors, and transforming many types of plants are well known. Examples of the production of transgenic plants having modified characteristics as a result of the introduction of a heterologous transgene include: U.S. Pat. No. 5,719,046 to Guerineau (production of herbicide resistant plants by introduction of bacterial dihydropteroate synthase gene); U.S. Pat. No. 5,231,020 to Jorgensen (modification of flavenoids in plants); U.S. Pat. No. 5,583,021 to Dougherty (production of virus resistant plants); and U.S. Pat. No. 5,767,372 to De Greve and U.S. Pat. No. 5,500,365 to Fischoff (production of insect resistant plants by introducing Bacillus thuringiensis genes).
In conjunction with such techniques, the isolation of plant R genes has similarly permitted the production of plants having enhanced resistance to certain pathogens. A number of these genes have been used to introduce the encoded resistance characteristic into plant lines that were previously susceptible to the corresponding pathogen. For example, U.S. Pat. No. 5,571,706 to Baker and Whitham describes the introduction of the N gene into tobacco lines that are susceptible to Tobacco Mosaic Virus (TMV) in order to produce TMV-resistant tobacco plants. WO 95/28423 describes the creation of transgenic plants carrying the Rps2 gene from Arabidopsis thaliana, as a means of creating resistance to bacterial pathogens including Pseudomonas syringae, and WO 98/02545 describes the introduction of the Prf gene into plants to obtain enhanced pathogen resistance. Cao et al. (1998) describes the introduction into Arabidopsis of the NPR1 cDNA expressed under the control of the 35S promoter to produce enhanced resistance to multiple bacterial pathogens.
The first R gene conferring virus resistance to be isolated from plants was the N gene of Nicotiana glutinosa tobacco (Whitham et al., 1994). The N gene (or homologs of this gene) is present in some but not all types of tobacco, and confers resistance to Tobacco Mosaic Virus (TMV). TMV is an important pathogen of not only tobacco, but also of other crop plants including tomato (Lycopersicon sp.) and pepper (Capsicum sp.). A review of the wide range of host species that serve as hosts to TMV is presented in Holmes (1946). TMV is the type virus of the genus Tobamovirus, which includes a number of closely related viral pathogens of commercially important plants. For example, the Tobamovirus group includes tomato mosaic virus, pepper green mottle virus and ondontoglossum ringspot virus, which is a pathogen of orchids (Agrios, 1997).
The N. glutinosa N gene is described in detail in U.S. Pat. No. 5,571,706 (xe2x80x9cPlant Virus Resistance Gene and Methodsxe2x80x9d) to Baker and Whitham, which is incorporated herein by reference. The sequence of this gene is available on GenBank under accession number U558886. U.S. Pat. No. 5,571,706 discloses the sequence of the N gene, as well as two cDNAs corresponding to the gene. The N gene (including the 5xe2x80x2 and 3xe2x80x2 regulatory regions) is over 12 kb in length and comprises five exons and four introns, encoding a full-length N protein of 1144 amino acids, with a deduced molecular mass of 131.4 kDa. cDNA-N is a cDNA encoded by the N gene; it is approximately 3.7 kb in length and encodes the full-length N protein. A second cDNA, cDNA-N-tr, is approximately 3.8 kb in length. It results from an alternative splicing pattern and encodes a truncated protein, N-tr, that is 652 amino acids in length and has a deduced molecular mass of 75.3 kDa. The N protein include TIR, NBS and LRR domains (Whitham et al., 1994, Baker et al., 1997). U.S. Pat. No. 5,571,706, and Whitham et al. (1994) describe the production of transgenic tobacco plants carrying a full-length N transgene; these plants show the HR response following TMV challenge.
While molecular genetic approaches that focus on individual R genes are promising, such approaches may be somewhat limited since each individual R gene will likely confer resistance to a relatively narrow range of pathogens. In contrast, exploitation of the SAR response could produce plants that are resistant to a broad range of pathogens of different types. The present invention is directed to a molecular genetic approach for producing plants that display an SAR response even in the absence of a plant pathogen, and which consequently show enhanced resistance to a broad spectrum of plant pathogens.
A molecular-genetic approach for producing a constitutive systemic acquired resistance (SAR) response in plants is provided. The SAR response in these plants is described as xe2x80x9cconstitutivexe2x80x9d since it occurs even in the absence of an infecting pathogen. Plants that exhibit this SAR response show enhanced resistance to a broad range of phytopathogens, including viruses such as TMV, bacteria and fungi. Among other things, the invention encompasses nucleic acid molecules that are used to produce plants that show such an SAR response, the plants themselves and methods of making such plants.
The invention is founded on the discovery that a non-pathogen induced SAR response is exhibited by transgenic plants that comprise the following elements:
(1) an R gene (or a cDNA encoding the R gene product(s)); and
(2) a transgene that expresses the R gene product (or an effective portion thereof).
Typically, element (1) is a native R gene, but it may also be a transgene comprising the R gene, a corresponding R cDNA, or a recombinant construct that otherwise expresses the R protein(s). (As described in more detail below, while most R genes have only a single reading frame and therefore encode a single R protein product, some R genes, including the tobacco N gene and the flax L6 gene, contain alternatively spliced exons, and therefore encode two R protein products.) The nucleic acid molecule of element (1) is, by itself, ordinarily sufficient to confer enhanced resistance to a pathogen that carries the corresponding Avr gene when expressed in an otherwise susceptible plant. Expression of this nucleic acid molecule is typically, but not necessarily, controlled by regulatory elements (promoter and terminator regions) that are associated with the native R gene.
Element (2) is typically a cDNA form of the R gene, and is generally expressed in the plant under the control of a promoter that produces expression levels that are greater than the native R gene promoter. High-level promoters suitable for use in this application are well known in the art, and include inducible and constitutive promoters. Inducible promoters may be employed in situations where it is advantageous to control expression of the non-pathogen induced SAR response. For example, it may be beneficial to turn on the SAR response once plants have reached a certain developmental stage, or when the likelihood of pathogen infection is highest. As described in more detail below, element (2) need not necessarily encode a complete R gene product but, in some circumstances, may encode only a portion of the R protein. The inventors have shown, for example, that element (2) may encode one or more domains of an R protein, but need not encode a complete R protein. For example, in certain embodiments, element (2) may encode all or part of a TIR domain of an R protein.
R genes and R gene products that may be employed in the invention include, but are not limited to, R genes encoding proteins having a TIR domain, such as: the N gene of tobacco (described in U.S. Pat. No. 5,571,706); the RPP5 gene of Arabidopsis (described in Parker et al., 1997) the RPP1 gene of Arabidopsis (described in Botella et al., 1998); the L6 gene of flax (described in Lawrence et al., 1995); and the M gene of flax (described in Anderson et al., 1997). Transgenic plants produced according to the invention (referred to as constitutive SAR plants for convenience) exhibit an SAR response and, as a result, shown enhanced resistance to a wide range of pathogens, including bacterial, viral and fungal pathogens.
In one embodiment of the invention, the invention provides transgenic plants comprising:
(1) a first nucleic acid molecule encoding one or more protein products of an R gene sufficient to confer resistance to a phytopathogen; and
(2) a second nucleic acid molecule encoding a protein comprising at least one polypeptide encoded by the R gene, wherein co-expression of the first and second nucleic acid molecules is sufficient to produce a systemic acquired resistance response in the plant.
The term xe2x80x9ca protein comprising at least one polypeptide encoded by the R genexe2x80x9d refers to a protein that includes at least one amino acid region (typically at least 10 contiguous amino acids) of the R protein. In other words, the polypeptide may be a sub-part of the R protein. Typically, such a polypeptide will be a domain of the R protein, such as a TIR domain, although less than an entire domain may also be employed.
The invention also encompasses a method of producing a plant that exhibits an SAR response in the absence of a pathogen infection. The method comprises introducing into a plant having an R gene a nucleic acid molecule that expresses a polypeptide component of a protein encoded by the R gene, wherein co-expression of the R gene and the polypeptide component produces an SAR response in the plant.
In another embodiment, the invention provides transgenic plants comprising a first nucleic acid molecule encoding an R gene product having a TIR domain, and a second nucleic acid molecule encoding a polypeptide comprising the TIR domain of the R gene product or an effective portion thereof, wherein co-expression of the first and second nucleic acid molecules produces a constitutive SAR response in the plant. In particular cases, the R gene product may be a product of a gene selected from the group consisting of the N gene of tobacco, the M and L6 genes of flax and the RPP5 and RPP1 genes of Arabidopsis.
In another embodiment of the invention, the R gene is the tobacco N gene, and the transgenic plants comprise the following elements:
(1) a first nucleic acid molecule encoding an N and an N-tr protein; and
(2) a second nucleic acid molecule encoding a protein comprising an N protein TIR domain.
The first nucleic acid molecule may be any molecule that encodes an N protein and an N-tr protein. Thus, for example, the first nucleic acid molecule may be a native N gene, an N transgene, or a cDNA-N/intron construct.
In one embodiment, the second nucleic acid molecule comprises cDNA-N or cDNA-N-tr. In another embodiment, the second nucleic acid molecule comprises a recombinant molecule encoding the N protein TIR region or a sufficient part of the TIR region to produce the SAR response when the first and second nucleic acid molecules are co-expressed in the plant.
Plants produced by the methods disclosed herein exhibit a constitutive SAR response and, consequently, enhanced broad-spectrum pathogen resistance. Parts of such plants, including seeds, fruit, stems, leaves and roots, may be utilized in conventional ways as food sources etc. The present invention is applicable to any plant type, but is expected to be particularly useful in plant types from which the corresponding R gene is obtained, and closely related plants. By way of example, application of the invention based on the tobacco N gene is expected to be particularly beneficial with respect to solanaceaous plants such as tobacco, tomato, potato and pepper.
The invention also encompasses methods for increasing or enhancing the disease resistance of plants. Such methods for increasing the disease resistance of plants involve plants having a first and second nucleic acid molecule as described supra. The methods comprise obtaining a plant having a first nucleic acid molecule encoding one or more protein products of an R gene sufficient to confer resistance to a phytopathogen; and then introducing into the genome of said plant a second nucleic acid molecule comprising at least one polypeptide encoded by the R gene, wherein co-expression of the first and second nucleic acid molecules is sufficient to produce a systemic acquired resistance response in the plant. The R gene of the first nucleic acid molecule can be native to the plant or can be introduced into the genome of the plant that lacks the R gene.